Figure 1. Knockdown of CUL3 or KCTD10 reduced the phosphorylation of epidermal growth factor receptor (EGFR) and HER2 through accumulation of RhoB-GTP in SKBR-3 cells.
(A) Western blots of SKBR-3 lysates, 72 h post-transfection with the indicated siRNAs. Rescue experiments of CUL3 and KCTD10 knockdown by infection with siRNA-resistant-FLAG-CUL3-, or siRNA-resistant non-tagged KCTD10-carrying lentivirus. pEGFR, phosphorylated EGFR (Tyr1068); pHER2, phosphorylated HER2 (Tyr1221/1222). The ratio of band intensities for pEGFR/EGFR or pHER2/HER2, normalized to control, was shown. (B, C) Western blots of SKBR-3 lysates, 72 h post-transfection with the indicated siRNAs. pEGFR, phosphorylated EGFR (Tyr1068); pHER2, phosphorylated HER2 (Tyr1221/1222). The ratio of band intensities for pEGFR/EGFR or pHER2/HER2, normalized to control, was shown. (D) SKBR-3 cells were transfected with the indicated siRNAs. Cell lysates were prepared and subjected to pull-down with Rhotekin-conjugated beads. Cell lysates and the pull-downed samples were analyzed by Western blot. (E) Western blots of SKBR-3 lysates, 72 h post-transfection with the indicated siRNAs. Cells were treated with C3 transferase (1 μg/ml) for 24 h before preparation of cell lysates. pEGFR, phosphorylated EGFR (Tyr1068); pHER2, phosphorylated HER2 (Tyr1221/1222). The ratio of band intensities for pEGFR/EGFR or pHER2/HER2, normalized to control, was shown. (F) SKBR-3 cells were treated with indicated siRNAs for 48 h. Trypsinized cells (total 0.5 × 105 cells) were then replated and treated with the same siRNA. Cell number was counted 72 h after replating. Data are mean ± SEM from three independent experiments. **P < 0.01. (G) Western blots of cell lysates of SKBR-3 cells treated with MLN4924 (1 μM) for 24 h. The asterisk indicates neddylated-CUL3 (Nedd8-CUL3). pEGFR, phosphorylated EGFR (Tyr1068); pHER2, phosphorylated HER2 (Tyr1221/1222). The ratio of band intensities for pEGFR/EGFR or pHER2/HER2, normalized to control, was shown. (H) Scheme of EGFR and HER2 phosphorylation regulated by the CUL3/KCTD10/RhoB axis. The CUL3/KCTD10 E3 complex constitutively ubiquitinates RhoB leading to its degradation. In CUL3 or KCTD10 knockdown cells, accumulated RhoB-GTP reduced the phosphorylation of EGFR and HER2, resulting in the inhibition of cell proliferation.
Source data are available for this figure.
